Swapping of Transmembrane Domains in the Epithelial Calcium Channel TRPV6

Home , TRPV6

bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Swapping of transmembrane domains in the epithelial calcium channel TRPV6

Appu K. Singh1*, Kei Saotome1* and Alexander I. Sobolevsky1

1Department of Biochemistry and Molecular Biophysics, Columbia University

650 West 168th Street, New York, NY 10032

*These authors contributed equally to this work

Correspondence and requests for materials should be addressed to A.I.S.

(Email: [email protected]; Tel: 212-305-4249; Fax: 212-304-5534)

Abstract

Tetrameric ion channels have either swapped or non-swapped arrangements of the S1-S4 and pore

domains. Here we show that mutations in the transmembrane domain of TRPV6 can result in conversion

from a domain-swapped to non-swapped fold. These results reveal structural determinants of domain

swapping and raise the possibility that a single ion channel subtype can fold into either arrangement in

vivo, affecting its function in normal or disease states.

Transient receptor potential (TRP) channels comprise a large and functionally versatile superfamily of

cation permeable ion channels that respond to a myriad of sensory modalities1. Structurally, TRP channels

are assemblies of four identical subunits arranged symmetrically around the pore axis2-6. The

transmembrane domain of TRP channels includes six transmembrane helices (S1-S6) and a pore loop (P-

loop) between S5 and S6. The first four helices (S1-S4 domain) form a bundle that plays the role of voltage

sensing domain in voltage-gated potassium channels7. S5, P-loop and S6 form the KcsA channel-like pore

domain, with the ion conduction pathway lined by the S6 helices and extracellular loops connecting the

P-loop helix to S5 and S6. In the cryo-EM reconstructions of the vanilloid subtype TRP channels TRPV12

and TRPV23,6, the S1-S4 domain of one subunit is packed against the pore domain of the neighboring

subunit, producing a domain-swapped arrangement similar to the voltage-gated potassium channel

chimera Kv1.2-2.17,8 .

TRPV6 and its close relative TRPV5 are TRP channels that are distinctly Ca2+-selective and play an

important role in calcium homeostasis9,10. Altered expression of TRPV6 is associated with numerous

human diseases, including various forms of cancer11. Accordingly, TRPV6 has emerged as an important

target for drug design12. We recently solved the structure of a TRPV6 construct engineered to produce

well diffracting crystals5. On the basis of electron density and cysteine crosslinking experiments, we built

a model of TRPV6 in which the S1-S4 domain of one subunit is packed against the pore domain of the

same subunit, creating a non-swapped architecture distinct from TRPV12 and TRPV23,6. Subsequent

studies of HCN13, CNG14, Slo115, Slo2.216 and Eag117 have revealed the prevalence of non-swapped

transmembrane domain architecture in tetrameric potassium channels.

Given the domain-swapped architectures of TRPV12 and TRPV23,6, and relatively high sequence

conservation in the transmembrane region (Supplementary Fig. 1), the absence of domain-swapping in

TRPV6 was a mystery that warranted further study. We initially wondered whether the mutations present bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

5 in the TRPV6 crystallization construct (TRPV6cryst) affected the domain arrangement. Compared to wild

type, TRPV6cryst has a 59-residue C-terminal truncation and four point mutations, including L495Q, which

resides in the S5 transmembrane helix (Supplementary Fig. 1). This L495Q substitution, which was a

spontaneous mutation that occurred during gene synthesis, was originally included in TRPV6cryst because

it expressed at 3-fold higher levels (Supplementary Fig. 2) than the same construct with the substitution

reverted to the native leucine (TRPV6*). To examine whether the distinct non-swapped arrangement of

TRPV6cryst resulted from the L495Q mutation, we were compelled to solve the structure of TRPV6*.

To crystallize TRPV6*, we used the same expression system, purification protocol and

5 crystallization conditions as for TRPV6cryst . TRPV6* crystallized in a nearly identical crystal lattice to

TRPV6cryst (Supplementary Table 1) and the overall shape of the molecule was very similar (Fig. 1). Despite

these similarities, to our great surprise, the structure of TRPV6* exhibited the domain swapped fold

observed in other TRP channels of known structure. Attempts to use the “non-swapped” TRPV6cryst model

for molecular replacement resulted in massive negative and positive signals in the Fo-Fc density maps.

Similar inconsistency between the crystallographic data and the model was observed when we attempted

to use the “swapped” molecular replacement model of TRPV6* to solve the structure of TRPV6cryst.

Therefore, the crystallographic data unambiguously demonstrates that the TRPV6* structure is domain-

swapped. Apart from the distinct connectivity between S4 and S5 as well as S6 and TRP helix, the domain-

swapped TRPV6* and non-swapped TRPV6cryst structures were almost identical. Superposition of TRPV6*

and TRPV6cryst excluding regions of S4-S5 (residues 464-497) and S6-TRP helix (residues 577-589) gave a

root mean square deviation (RMSD) of 0.865 Å.

We reproduced crystals of TRPV6cryst and confirmed its non-swapped conformation. Notably, we

were able to collect new crystallographic data, where both the S4-S5 and S6-TRP helix connectivity in

TRPV6cryst is unambiguously resolved in the 2Fo-Fc and Fo-Fc omit maps (Supplementary Fig. 3), in stark bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

contrast to our previous data5, where density for thirteen residues in S4-S5 linker (F471-Q483) was missing.

Similarly, both the 2Fo-Fc and robust Fo-Fc omit maps for TRPV6* demonstrated completely different

connectivity of transmembrane domains (Supplementary Fig. 3) compared to TRPV6cryst. These structural

data therefore suggest that the single leucine L495 to glutamine substitution in S5 is responsible for the

absence of domain swapping in TRPV6cryst. Because the sequence of TRPV6* is closer to wild-type, the

domain-swapped architecture of TRPV6 likely represents the physiologically relevant domain

arrangement.

The presence or absence of transmembrane domain swapping could impact the functional

behavior of TRPV6 considerably. Nonetheless, ratiometric fluorescence measurements of cells loaded

2+ with the Ca sensitive dye Fura-2 showed that the three constructs, TRPV6*, TRPV6cryst and wild type

TRPV6, demonstrate pore permeability to Ca2+ and ion channels block by Gd3+ (Fig. 2). Indeed, the pore

architectures of TRPV6cryst and TRPV6* are indistinguishable (Supplementary Fig. 4a-b), and anomalous

signals for Ca2+ and Gd3+ indicated that the positions of the pore cation binding sites are similar in TRPV6*

5 (Supplementary Fig. 4c-f) and TRPV6cryst . Thus, the structural and functional integrity of the TRPV6 pore

is overall preserved in both domain-swapped and non-swapped folds. Despite these gross similarities,

kinetic analysis indicated the rate of increase in Fura-2 signal is faster in TRPV6* compared to TRPV6cryst

(Supplementary Fig 5). Therefore, the Ca2+ entry function is somewhat compromised in the non-swapped

TRPV6cryst construct, presumably due to differences in allosteric communication between the pore domain

and the rest of the protein.

How does the L495Q mutation result in the non-domain swapped fold of TRPV6? In TRPV6*, L495

projects its side chain into a hydrophobic pocket formed by residues of S5 and S6 of one subunit and

residues at the S4-S5 elbow of the adjacent subunit (Fig. 1e). By contrast, in TRPV6cryst, Q495 is rotated by

~120⁰ with respect to the orientation of L495 in TRPV6* and forms polar interactions with the hydrophilic bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

pocket formed by S4-S6 and the TRP helix of the same subunit (Fig. 1f). These observations suggest that

hydrophobic burial of the L495 residue plays an important role in driving the domain-swapped fold of

TRPV6. Disruption of these hydrophobic interactions leads to the non-swapped fold. In other members of

TRPV family, either leucine or valine occupy the position homologous to L495 in TRPV6 (Supplementary

Fig. 1b). Further, a hydrophobic pocket similar to TRPV6 is also present in the structures of TRPV1/22,3,6,

suggesting a conserved role for this region in determining the domain arrangement in TRPV channels.

In the case of non-swapped potassium channels, the absence of domain-swapping was attributed

to shorter S4-S5 linkers13-17. We tested whether TRPV6 domain arrangement could be biased toward a

non-swapped conformation by shortening of the S4-S5 linker, which has a well-defined helical structure

in the domain-swapped TRPV6* but lacks secondary structure in TRPV6cryst. Remarkably, TRPV6*

constructs with up to ten residues deleted in the S4-S5 linker expressed robustly and could be isolated as

a monodisperse species with gel filtration elution time corresponding to the tetrameric assembly

(Supplementary Fig. 6a-b). We determined the crystal structure of TRPV6* with four residues deleted in

the S4-S5 linker (TRPV6*-del1) and confirmed its non-swapped architecture (Supplementary Fig. 6c-d).

Therefore, the length of the S4-S5 linker is indeed a critical determinant of domain-swapping.

Interestingly, ratiometric fluorescence measurements did not detect measurable Ca2+ influx in HEK cells

expressing TRPV6*-del1, suggesting that while the tetrameric expression and assembly in this mutant is

preserved, gating function is significantly perturbed.

In summary, our studies demonstrate that small sequence changes can result in a major alteration

of the transmembrane domain arrangement of a tetrameric ion channel. Depending on the nature of the

mutation, this structural alteration can lead to strong or weak changes in ion channel function. We

speculate that a single tetrameric ion channel subtype can adopt swapped or non-swapped

transmembrane domain arrangements in vivo, depending on the presence of mutations, post- bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

translational modifications, or other perturbations. The impact of changes in domain-swapping on ion

channel function may play a role in pathophysiology.

Figure 1. Non-swapped domain arrangement as a result of single residue substitution. a-b, Extracellular

views of TRPV6* (a) and TRPV6cryst (b), with four subunits (A-D), each shown in different color. Note, the

peripheral S1-S4 domains are adjacent to the S5-S6 pore domains of the same subunit in TRPV6cryst, while

the adjacent domains are from neighboring subunits in TRPV6*. c-d, Side views of one S1-S4 domain and

two S5-S6 pore domains illustrating S4-S5 and S6-TRP helix connectivity unique to swapped (c) and non-

swapped (d) architectures. e-f, Close-up views of the regions surrounding L495 in TRPV6* (e) and Q495 in

TRPV6cryst (f).

Figure 2. Ca2+ permeability and Gd3+ block are preserved in TRPV6 constructs with distinct domain

arrangements. a-b, Representative ratiometric fluorescence measurements for HEK cells expressing

TRPV6*. Arrows indicate the time at which the corresponding ion was added. After resuspending the cells

in nominally calcium-free buffer, addition of Ca2+ resulted in robust concentration-dependent increase in

Fura-2 signal (a). In contrast, pre-incubation of cells with increasing concentrations of Gd3+ resulted in

concentration-dependent reduction in Fura-2 signal (b). c-d, Normalized changes in F340/F380 at varying

2+ 3+ concentrations of Ca (c) and Gd (d) calculated for wild type TRPV6 (blue), TRPV6cryst (red) and TRPV6*

3+ (green). In d, ΔF340/F380 was measured after preincubation with Gd and subsequent addition of 2 mM

Ca2+.

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Methods

Constructs

5 Our previous crystallization construct TRPV6cryst comprised of residues 1-668 of rat TRPV6 and contained

the point mutations I62Y, L92N, M96Q and L495Q. We made a new construct TRPV6*, which is identical

to TRPV6cryst except the reversal of the single L495Q mutation in S5 to the native leucine residue. To

understand the role of S4-S5 linker in domain swapping, TRPV6*-del1, TRPV6*-del2, TRPV6*-del3 and

TRPV6*-del4 constructs (Supplementary Fig. 6a) were prepared by deleting four (477-480), six (475-480)

eight (473-480) and ten (471-480) residues in S4-S5 linker, which were disordered in the original TRPV6cryst

structure5.

Expression and Purification

TRPV6 constructs described in this paper were expressed and purified as previously described for the

5 TRPV6cryst . In brief, TRPV6 constructs were introduced into the pEG BacMam vector with C-terminal

thrombin cleavage site (LVPRG) followed by eGFP and streptavidin affinity tag (WSHPQFEK), for the

expression in suspension of baculovirus-transduced HEK293 GnTI- cells. 48-72 hours after transduction

with P2 virus, cells were harvested, washed with PBS at pH 8.0 and after sonication, cellular membranes

were prepared. The protein was extracted from cellular membranes using 20 mM n-dodecyl-β-D-

maltopyranoside, and purified using Strep-Tactin affinity chromatography followed by size exclusion

chromatography.

Crystallization and structure determination

Optimized crystals of purified TRPV6* or TRPV6*-del1 were grown in the same condition as crystals of

5 TRPV6cryst , which included a reservoir solution consisting of 20-24% PEG 350 MME, 50 mM NaCl and 50

mM Tris-HCl pH 8.0-8.5 in hanging drop configuration at 20⁰C. Prior to crystallization using hanging drop

method, the protein was subjected to ultracentrifugation (Ti100 rotor, 40000 rpm, 40 min, 4C) to remove bioRxiv preprint doi: https://doi.org/10.1101/141523; this version posted May 24, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

the aggregated protein. Crystals were cryoprotected by serial transfer into the buffer composed of 100

mM NaCl, 100 mM Tris-HCl pH 8.2, 0.5 mM DDM and 50 mM ammonium formate, containing increasing

concentration of PEG 350 MME, with maximum concentration of up to 33-36%, and then flash frozen in

2+ 3+ liquid nitrogen. To obtain crystals with Ca or Gd , protein was incubated with 10 mM CaCl2 or 1 mM

GdCl3, respectively, for at least 1 hour at 4˚C prior to crystallization.

X-ray diffraction data collected at APS (beamlines 24-ID-C/E), NSLSII (beamline 17-ID) or ALS

(beamline 5.0.2) were indexed, integrated and scaled using XDS18 or HKL200019. The initial phase

information and structures were solved by molecular replacement using Phaser20 and the structure of rat

5 TRPV6cryst (PDB ID: 5IWK) as a search probe. Most of TRPV6*, including the ankyrin domain, S1-S4, pore

module and C-terminal hook, was nearly identical to the TRPV6cryst; the rest of the structure was built

using the omit map as a guide. The robust electron density for the S4-S5 linker was evident from the initial

phases obtained by molecular replacement, and map features improved further during refinement. The

model was refined by alternating cycles of building in COOT21 and automatic refinement in Phenix22 and

Refmac23. All structural figures were made using PyMOL23 (www.pymol.org)

Fura 2-AM measurements

2+ The intracellular Ca measurements from HEK cells expressing rat TRPV6 or TRPV6cryst or TRPV6* were

performed as described previously5. All Fura2-AM-based fluorescence measurements were conducted

using spectrofluorometer QuantaMasterTM 40 (Photon Technology International) at room temperature in

a quartz cuvette under constant stirring.

Supplementary Figure 1. Topology and sequence alignment of TRPV subunits. a, Membrane topology of

TRPV subunits. Red circles and red line indicate four point mutations and C-terminal deletion in TRPV6cryst

construct, respectively. b, Sequence alignment of the transmembrane domain regions in rat TRPV

subtypes. Helices are indicated by cylinders above the sequence. L495 (open red box) and the selectivity

filter in TRPV6 (thick red line) are highlighted. ¥ marks the N-linked glycosylation site in the extracellular

loop connecting S1 and S2 conserved in TRPV6 (and TRPV5) channels.

Supplementary Figure 2. FSEC traces for crudely solubilized TRPV6cryst and TRPV6*. FSEC traces were

recorded using Superose 6 size-exclusion chromatography column monitoring GFP fluorescence. Note the

difference in the peak height, indicating approximately 3 fold greater expression levels of TRPV6cryst

compared to TRPV6*.

Supplementary Figure 3. Dramatic change in TRPV6 transmembrane domain connectivity as a result of

single-residue substitution in S5. a-d, Transmembrane portions of TRPV6* (leucine at position 495, left)

and TRPV6cryst (glutamine at position 495, right) viewed parallel to the membrane. S4-S5 (upper row) and

S6-TRP helix (bottom row) connectivity is illustrated by 2Fo-Fc (blue mesh, 1) and omit Fo-Fc (yellow

mesh, 2.5) maps.

Supplementary Figure 4. TRPV6 structures with swapped and non-swapped transmembrane domains

have identical ion channel pores. a-b, Superposition of the pore domains in TRPV6* (cyan) and TRPV6cryst

(yellow) viewed parallel to the membrane (a) or extracellularly (b), with residues important for cation

binding shown in stick representation. Calcium atom is shown as a green sphere. c-f, Side (c,e) and top

(d,f) views of the TRPV6* pore. Front and back subunits in c and e are removed for clarity. Green and pink

mesh shows anomalous difference electron density for Ca2+ (c-d, 3) and Gd3+ (e-f, 3.5), respectively,

and ions are shown as spheres of the corresponding color. Positions of cation binding sites are similar to

5 TRPV6cryst .

Supplementary Figure 5. Different kinetics of ratiometric curves for TRPV6* or TRPV6cryst at

physiological extracellular calcium concentration. a-b, Representative ratiometric fluorescence

measurements for HEK cells expressing TRPV6* (a) or TRPV6cryst (b). Arrows indicate the time at which 2

mM Ca2+ was added. The curves were fitted with one (red) or two (blue) exponentials. Note, TRPV6*

kinetics was well described by the double but not the single exponential. Over three measurements, the

time constants of the single exponential fit (single) were 14.5 ± 2.7 s for TRPV6* and 110 ± 16 s for

TRPV6cryst, fast = 2.2 ± 1.0 s, slow = 54 ± 11 s and Afast = 0.57 ± 0.01 for TRPV6* and fast = 5.4 ± 2.8 s, slow =

135 ± 23 s and Afast = 0.10 ± 0.03 for TRPV6cryst. At n = 3, the values of single and Afast for TRPV6* and

TRPV6cryst were statistically different (t-test, P < 0.05).

Supplementary Figure 6. Deletions in S4-S5 preserve TRPV6 tetrameric assembly and generate non-

swapped topology. a, Sequence alignment of TRPV6* and mutants with deletions in the S4-S5 linker.

Leucine L495 is indicated by the red circle. The sequence corresponding to the resolved regions in TRPV6*-

Del1 structure is highlighted by yellow. b, FSEC traces for purified TRPV6* without and with deletions in

S4-S5 following tryptophan fluorescence. Note, all samples demonstrate a single monodisperse

tetrameric peak. c, Transmembrane domain of a single TRPV6*-Del1 subunit demonstrates a non-

swapped architecture with the characteristic S6-TRP helix connectivity. Blue mesh shows 2Fo-Fc map (1).

d, Close-up view of the S4-S5 illustrating boundaries of a missing region (residues 471-485).

Supplementary Table 1. Data collection and refinement statistics.

2+ 3+ TRPV6* TRPV6cryst TRPV6*-Del1 TRPV6*-Ca TRPV6*-Gd

Data Collection Beamline APS-24ID-C APS-24ID-C APS-24ID-C APS-24ID-C APS-24ID-C

Space group P4212 P4212 P4212 P4212 P4212 Cell dimensions 146.03 145.53 145.32 145.48 144.02 a, b, c, (Å) 146.03 145.53 145.32 145.48 144.02 116.05 114.13 113.67 110.50 114.34

α, β, γ (⁰) 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 Wavelength (Å) 0.9791 0.9791 0.9791 1.75 1.00 Resolution (Å)* 50.0 - 3.25 50.00 - 3.45 50.0 - 3.40 50.0 – 3.6 50.0 - 3.85 (3.44 - 3.25) (3.56 - 3.45) (3.52- 3.40) (3.68 - 3.60) (3.99 - 3.85) Completeness 98.4 99.8 99.26 99.5 95.9 (%)* (97.2) (99.6) (95.13) (95.1) (91.8) Redundancy* 8.6 8.5 8.5 18.9 8.3 (7.1) (8.5) (6.7) (17.3) (5.5) |I/σ|* 18.31 11.3 11.44 11.9 12.1 (1.05) (1.1) (1.22) (1.07) (1.10)

Rmeas (%)** 8.7 9.93 13.0 14.6 19.4 (190.9) (194.45) (140.9) (260.5) (255.8)

CC1/2 99.8 99.6 99.9 99.6 99.5 (60.3) (44.65) (61.8) (67.1) (42.9) Refinement

Resolution (Å)* 50.0 - 3.25 49.91 - 3.45 50.00 - 3.40 50.0 – 3.6 50.0 - 3.85 (3.36 - 3.25) (3.66 - 3.45) (3.52-3.40) (3.68 - 3.60) (3.99 - 3.85) Completeness 96 99.8 99.26 96 97.86 (%) (93.8) (98.5) (95.13) (93.8) (99.20) Number of 18531 17758 17211 18531 20034 reflections (1724) (791) (1605) (1724) (1973)

Rwork/Rfree 0.273/0.297 0.278/0.298 0.286/0.305 0.273/0.297 0.275/0.295 Number of atoms Total 4722 4809 4727 4722 4897 Ligand 16 3 16 16 155 B-factor (Å2) Protein 120.5 147.07 145.47 120.5 150.11 Ligand 77.27 86.12 164.66 77.27 164.71 RMS deviations Bond length (Å) 0.003 0.003 0.009 0.003 0.002 Bond angles (⁰) 0.7 0.84 0.84 0.7 0.51 Ramachandran Favored (%) 94.6 93.6 94.6 94.6 94.6 Allowed (%) 5.4 5.9 4.9 5.4 4.8 Disallowed (%) 0.0 0.5 0.5 0.0 0.6

*Highest resolution shell in parentheses. 5% of reflections were used for calculation of Rfree.